Prediction of the binding interactions between rosmarinic acid and cysteinyl leukotriene receptor type 1 by molecular docking and immobilized receptor chromatography

Drug–protein interaction analysis is still at the center of research efforts to illustrate binding mechanisms and provide valuable information for selecting drug candidates with ideal properties in the early drug discovery stage. We present the prediction of the binding of rosmarinic acid (RA) to cysteinyl leukotriene receptor type1 (CysLTR1) by molecular docking. According to our findings, CysLTR1 is a potential anti-inflammatory target of RA. Under this assumption, we prepared the immobilized CysLTR1 column via a one-step method and characterized the immobilized CysLTR1 by fluorescent and chromatographic analyses. Furthermore, we used the immobilized CysLTR1 column to evaluate the binding interactions between RA and the immobilized receptor. Molecular docking showed that Tyr 249, Phe 174, Thr 280, Pro 177, and Thr 100 are the main sites for RA to interact with CysLTR1. The main forces that drive the findings are hydrogen bonds and hydrophobic interactions. Characterization results show that CysLTR1 is successfully immobilized with high specificity and stability. Almost no non-specific binding is observed on the immobilized CysLTR1 gels. The association constant and the binding sites are calculated to be 7.268 × 105 L mol−1 and 1.237 × 10−8 mol L−1 by injection amount-dependent method. These results, taken together, confirm the potential target of RA on the anti-inflammatory effect. We believe that it can provide valuable reference information on the in-depth exploration of drug–protein interaction mechanisms, and lead compound screening by this method.


Introduction
6][7][8][9] Thus, increased attention has been drawn to the essential role of binding affinity investigations between drugs and proteins in discovering lead compounds.
During the past few years, dramatic changes have taken place in evaluating drug-protein interactions due to the rapid development of detection technology, biotechnology, as well as instrumental technology.2][23] These methods are simple to operate and do not require the separation of proteins and drugs, however, they have high requirements for the sample purity.As an alternative, capillary electrophoresis has contributed a lot to exploring the binding interactions with low consumption of samples.Notably, the calculation of mathematical formulas needs more attention because it always varies when obtaining binding parameters.Moreover, ongoing work is needed to break their limitations in long analysis time, lacking sufficient sensitivity, relatively poor selectivity, and stability.
Chromatographic methods, especially receptor chromatography, have been popularized in revealing the binding interactions between drugs and receptors. 24,25It has enabled fast analysis and specic recognition into a single analysis of the analyte, which merits the method's high speed, specicity, stability, and throughput.Thus, it is superior to the conventional methods in elds, such as obtaining drug-protein interaction parameters with high precision and reproducibility.For such purposes, frontal analysis and zonal elution were mainly used to calculate the binding parameters.However, both two methods suffer from the deciencies of long analytical periods and a large amount of sample consumption.To overcome the shortcomings of the two methods, Zhao et al. developed an injection-amount dependent method and non-linear chromatography for fast analysis of the binding interactions between drugs and the immobilized receptors. 24,26][28] The results showed high consistency with other classical methods in the literature.
Rosmarinic acid (RA), a water-soluble phenolic compound, has been discovered in species of the Boraginaceae family and the Nepetoideae subfamily of the Lamiaceae family.0][31] This implies that RA has the potential to be a specic ligand of cysteinyl leukotriene receptor type 1 (CysLTR1).Herein, this work aimed to simulate the binding interaction between RA and CysLTR1 by molecular docking.Ongoing work was performed to validate the application of the immobilized CysLTR1 column in exploring the binding parameters by the injection-amount-dependent method.We believe that this strategy is promising for predictions and investigations of the bindings for other functional proteins and drugs.
The centrifuge, with a Cat.No. of Sorvall LYNX 4000, was used to separate the supernatant.The precipitant of the cell lysate was obtained from ThermoFisher Scientic (China) Co., Ltd.The ultrasonic cell crusher (Cat.No. JY92-IIN) was bought from Ningbo Xinzhi Biotechnology Co., Ltd, which was used to disrupt the cells that produced the target proteins.Furthermore, we used a ZZXT-A packing machine purchased from Dalian Elite Analytical Instrument Co., Ltd to prepare the immobilized CysLTR1 column.Chromatographic experiments were performed by the Shimadzu LC-2030 high-performance liquid chromatography apparatus with an isocratic pump, an autosampler, a column temperature chamber, and a UV detector (Shimadzu Inc., Japan).

Molecular simulations
The crystal structure of CysLTR1 was downloaded from the Protein Data Bank (PDB ID: 6RZ5).We kept the receptor in rigid mode for the simulation process.The pre-bonded small ligands, such as zarlukast, water, and ions, were removed by PyMOL.The crystal structure was pre-treated by adding polar hydrogens and Gasteiger charges before docking.For the three drugs and the screened bioactive compound, the structures were generated by ChemDraw Ultra 8.0.A rectangular box enclosing the ligand-binding site of zarlukast in CysLTR1 was applied as the sampling space for docking.The size for the grid box was set as 60 Å × 60 Å × 60 Å.We performed three independent docking runs and selected the binding pose with the lowest docking score.We analyzed and visualized the receptorligand complex using Discovery Studio 4.5.

Expression of Halo-tagged CysLTR1
We transformed the fused Halo-tagged CysLTR1 plasmid into Escherichia coli (E.coli) BL21(DE3) cells by the heat-shock method.The cells were then spread onto a 25 mL Luria-Bertani (LB) agar plate and incubated overnight at 37 °C with ampicillin at a nal concentration of 100 mg mL −1 .The next day, we picked up a single colony and transferred it into 150 mL of LB medium for another round of growth at 37 °C under an ampicillin atmosphere.Aer that, the cells were transferred into an auto-induction medium for expression of the target protein at 37 °C for 10 hours.Finally, we harvested 8.5 g of the cells by centrifugation at 4 °C for 20 min with a rotating speed of 7000 rpm.The harvested cells were then resuspended in 20 mM phosphate buffer (PB, pH = 7.40), disrupted, and centrifuged at 4 °C for 30 min.All the samples were then collected for further use.

Immobilization of CysLTR1
Using a conventional construction process from the literature, 32 we prepared the stationary phase by the following procedures: (1) activation of the silica gel.Briey, 1.0 g of dried aminopropyl silica gel was weighed and transferred into a ask.The gels were then suspended in 10.0 mL of dimethylformamide (DMF) within 82.98 mg of 6-chlorohexanoic acid (1.2 eq.) and 240 mL of N,N-diisopropyl ethylamine (3.0 eq.).Sequentially, 210 mg of 2-(7-azabenzotriazol-1-yl)-N,N,N 0 ,N 0 -tetramethyluronium hexa-uorophosphate was added into the ask, and the reaction was allowed to occur for 2.0 hours at ambient temperature.Aer that, the gel was ltered and sequentially rinsed with DMF and 20 mM phosphate buffer (PB, pH = 7.40).( 2) Immobilization of CysLTR1.The gels were re-suspended into 50 mL of the supernatant of the cell lysate containing the expressed CysLTR1.The gels were then reacted for 1.0 h to prepare the immobilized receptor.The immobilized CysLTR1 was then packed into the stainless-steel column (4.6 × 30 mm) using 20 mM PB (pH = 7.40) as slurry and propulsive agent under the pressure of 400 bar for 1 hour.

Characterization of the immobilized CysLTR1
Fluorescent analysis.We prepared the bare silica gel, aminofunctionalized gel, 6-chlorohexanoic acid modied gel, and CysLTR1 coated gel through the aforementioned procedures.200 mg of these gels were incubated with 1 mL of cyanine5 maleimide for 1.0 h.The gels were then washed with 20 mM PB (5.0 mL × 3) and examined under the uorescence microscope with an excitation wavelength of 670 nm.
Chromatographic analysis.Twenty millimolar PB (pH = 7.40) was taken as the mobile phase.We injected four drugs into the immobilized CysLTR1 column to test their binding behaviors on the immobilized CysLTR1 column.The injection volume and the ow rate were set to 10 mL and 0.2 mL min −1 .The detection wavelengths were 262 nm for pranlukast, 242 nm for zarlukast, 238 nm for MK-571, and 205 nm for sodium nitrite, respectively.Isocratic elution mode was performed during the immobilized CysLTR1 chromatography with 20 mM PB (pH = 7.40) as the elution buffer throughout the elution process.The ow rate was set the same as before.Taking zarlukast as the probe, we tested the stability of the chromatographic system for 30 days.

Binding interaction investigations
We applied the injection amount-dependent method and nonlinear chromatography to investigate the binding interactions between the drugs and the immobilized CysLTR1 at 37 °C.We prepared the stock solution in a nal concentration of 5 mM for each drug.We prepared the analyte by diluting the stock solutions with 20 mM PB (pH = 7.40).The concentrations were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM for zarlukast; 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM for pranlukast; 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM for MK-571, and 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 1.1 mM for RA.Aer passing through the 0.45 mm lter membrane, we loaded the drugs onto the column containing the immobilized CysLTR1 in triplicates.The injection volume and the ow rate were set at 10 mL and 0.6 mL min −1 .The wavelengths detected for the three drugs were consistent with the aforementioned ones, while for RA, it was 330 nm.The mobile phases were 50 mM PB in 2-propanol (91 : 9, V : V) for pranlukast and zarlukast; it was 20 mM PB (pH = 7.40) for MK-571 and RA, respectively.We employed isocratic elution to investigate the binding parameters between the drugs and immobilized CysLTR1.The elution solutions and the ow rate we used here were the same as the ones mentioned before.All the chromatographic proles were recorded, and the data were analyzed.

Molecular docking analysis
As reported in the literature, the ligand binding pocket of CysLTR1 extends from the extracellular loop2(ECL2) across the receptor toward a gap between the transmembrane helices, 4 (TM4) and 5 (TM5), then deep into the middle part of the TM 7. 33 Tyr 104 and Tyr 249 residues that are located at the ECL2 sections are demonstrated to create polar interactions with the ligand.Arg 79 on the side chain is proven to participate in the formation of salt bridges and is involved in hydrophobic interactions with the ligands.Furthermore, some amino acids in the CysLTR1, i.e., Phe 158, Tyr 108, Ser 155, Leu 189, His 190, Arg 253, His 256, Pro 176, Val 277, Thr 100, and Leu 281, were involved in the binding pockets and were identied as major sites for the binding of ligands by hydrogen bonds and hydrophobic interactions.
The chemical structures, 3D and 2D overviews of the molecular simulations, between RA, pranlukast, zarlukast, MK-571, and CysLTR1 are displayed in Fig. 1.The results revealed that specic interactions occurred between RA and CysLTR1.The amino residues, Phe 174, Thr 280, Pro177, and Tyr 249 on CysLTR1 participated in the formation of hydrogen bonds with RA.While Leu 281, Arg 253, Val 277, and Thr 100, mainly interacted with RA by hydrophobic interactions through the benzene ring of RA.Meanwhile, we found that hydrogen bonds, van der Waals force, and hydrophobic interactions were the main driving forces for pranlukast, zarlukast, and MK-571 to bind to CysLTR1.The residues were located all around the binding pocket of the receptor.Taken together, these results demonstrated that the specic binding sites of RA to CysLTR1 were consistent with the results from the literature, reminding us that RA is a potential anti-inammatory ligand of CysLTR1.

Expression of Halo-tagged CysLTR1
As a fundamental step, the expression of the target proteins plays a pivotal role in the preparation of functional protein columns for subsequent investigations, especially regarding the largest trans-membrane protein family members: G proteincoupled receptors.In this work, we expressed the Halo-tagged CysLTR1 by a previously reported method. 34A prokaryotic system, Escherichia coli (E.coli) cell, was used to express the target protein because it benets the method of low cost and fast growth rate.Briey, a heat-shock method was used to transform the fused Halo-tagged CysLTR1 plasmid into the E. coli BL21 (DE3) cells.According to the conventional target protein expression manual, the Halo-tagged CysLTR1 was induced in an LB medium at different temperatures using varied concentrations of isopropyl-beta-D-thiogalactopyranoside (IPTG) as the inducer.However, the outcome was not satisfying because the target protein cannot be expressed very well by using such a method.We attributed this result to the low stability and the aggregation of the recombinant protein by using the methods mentioned above.As an alternative, the auto-induction medium was applied to replace the conventional IPTG-based induction due to its simple and soluble expression. 35,36When the target recombinant protein is in a soluble and biologically active form, the inefficient refolding of the protein can be ignored, and thus, it has been proven to be advantageous to use the target protein as a template.
Fig. 2 shows sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the expressed Halotagged CysLTR1.We found that a new band appeared in the auto-induction medium located at the position corresponding to the molecular weight between 55 kDa and 75 kDa.By calculating the molecular weight of the newly appeared band, we determined it to be Halo-tagged CysLTR1 as the value highly agreed well with the theoretical molecular weight of the protein.
From this point of view, we believe that the target protein has been successfully expressed.Meanwhile, we found that in comparison with the supernatant, this new band was rarely expressed in the precipitant.Even if the loading volume increased to 20 mL, there were no signicant changes.We considered that this outcome is rational because the target protein is mainly expressed in soluble forms in the supernatant, therefore, it can retain its bioactivity.Thus, the content of the target band in the precipitant is very low.In brief, these ndings indicate that the recombinant protein is successfully expressed in the supernatant of the cell lysate.In this work, we immobilized CysLTR1 through the specic interaction between haloalkane dehalogenase and 6-chlorohexanoic acid (Fig. 3).The content of the target protein was determined by analyzing the difference in grayscale of the target protein band before-and aer-immobilization by SDS-PAGE (Fig. 1).The content of the immobilized receptor percentage was calculated to be 3.86% (before) and 1.42% (aer) by using a band as the internal standard.In this way, the amount of the immobilized CysLTR1 was calculated to be 18.42 mg g −1 .Furthermore, we used uorescent analysis and chromatographic investigations to test whether CysLTR1 was successfully immobilized onto the gel surface.
Fluorescent analysis.Fluorescent analysis is a frequently used method to detect biomolecules due to their high sensitivity, good reproducibility, high specicity, and ease of use. 37,38It usually employs uorophore-labeled materials, or specic antibodies to detect the concentration of the target bio-molecule using the concentration of the uorescence intensity.Beneting from the advantages of uorescent analysis, it has been popularized in elds, such as chemistry, materials science, physical, medical, environmental, and biotechnological applications.Some modern techniques, such as uorescence sensors, have been developed to detect biomolecules, such as uorescent sensors, uorescence microscopy, and enzyme-assisted uorescence techniques.In this set of experiments, we intend to detect the immobilized CysLTR1 based on the uorescent method.
Fig. 4 displays the bare silica gel, amino-functionalized silica gel, 6-chlorohexanoic acid-modied gel, and CysLTR1-coated Fig. 2 SDS-PAGE analysis of CysLTR1.Lane M: protein marker, lane L 1 , L 2 : LB medium, lane A 1 , A 2 : auto-induction medium, lane S 1 , S 2 : supernatant of the cell lysate, lane P 1 , P 2 : precipitant of the cell lysate.Lane A: supernatant of the cell lysate after immobilization, lane B: supernatant of the cell lysate before immobilization.The subscript 1 and subscript 2 referred to the loading volume of the analyte as 10 mL and 20 mL.
Fig. 3 Immobilization of CysLTR1 via one-step relying on the specific interaction between haloalkane dehalogenase and 6-chlorohexanoic acid.
gel incubated with a specic substrate-cyanine 5 maleimide for 1.0 h.Cyanine 5 maleimide is a kind of dye that has been used to label biological molecules for uorescence imaging and other uorescence-based biochemical analyses.It is usually recommended for labeling thiol groups of the molecules.Herein, we used it to label the immobilized CysLTR1 onto the surface of the silica gel.We found that rare uorescent signals appear in the bare silica amino-functionalized silica gel, as well as 6chlorohexanoic acid-modied gel.Compared with these samples, CysLTR1-modied silica gel showed signicant it can be labeled, while the other three groups could not be labeled.Thus, we believed that Halo-tagged CysLTR1 was successfully immobilized onto the gel surface.Chromatographic analysis.We investigated the specicity of the immobilized CysLTR1 column by testing the capacity factors of pranlukast, zarlukast, and MK-571 on the column with the values on the control column, which was packed with bare silica gel as the stationary phase.No substantial differences were observed in the capacity factors for these columns, suggesting that nonspecic interactions rarely occurred between the drugs and the immobilized CysLTR1.The specicity of the column containing the immobilized CysLTR1 was tested by determining the chromatographic proles and the retention times of the three drugs on the immobilized CysLTR1 column.The representative chromatographic proles of sodium nitrite, pranlukast, zarlukast, and MK-571 are depicted in Fig. 4. We found that the four drugs presented different retention times on the immobilized CysLTR1 column.Meanwhile, the retention times of the three drugs, pranlukast, zarlukast, and MK-571, were all longer than the retention time of sodium nitrite.This result is reasonable because sodium nitrite is a non-specic ligand of CysLTR1.Thus, we attributed the retention time of sodium nitrite to the void time of the chromatographic system.In addition, the other three drugs presented longer, but varied, retention times than sodium nitrite.Retention times were 0.298 min for sodium nitrite, 5.281 min for zarlukast, 7.234 min for MK-571, and 8.358 min for pranlukast, respectively.This result is rational as the three drugs behaved as specic ligands of CysLTR1.From this aspect, we reasoned that variation in the retention times conrmed that the immobilized receptor still retained specicity for its ligands aer immobilization and packing into the stainless-steel column.
Furthermore, the stability of the immobilized CysLTR1 column was tested using MK-571 as a probe.We tested it by injecting the drug into the column for 30 consecutive 30 days.The representative chromatograms of MK-571 on the immobilized CysLTR1 column are depicted in Fig. 4F.The results demonstrate that no observable difference emerged in either the chromatographic proles or the retention times of the drug.It proved that the column containing the immobilized CysLTR1 can be stable for at least 30 days.The column could be re-used for as many as 900 injections.The relative standard deviation of the retention times for MK-571 was 0.5%.In this case, the immobilized CysLTR1 proved to be stable for at least 30 days with a sampling efficiency of 900 injections.Such results provide evidence of the stability of the column.Taken together, the immobilized CysLTR1 column was proved to be stable for at least one month.

Binding interaction analysis
The binding and elution of the drug on the immobilized receptor chromatography is similar to the absorption and desorption processes in vivo.Thus, we studied the immobilized CysLTR1 to investigate the binding interaction between drugs and the receptor.In this situation, isocratic elution was used to allow a single mobile phase to be used for both sample application and elution in the immobilized CysLTR1 column.The elution buffer we used was the same as the mobile phase to illustrate the binding interactions by the injection-amountdependent method.It is believed to be a convenient and reliable method that was rst proposed in 2014.Unlike frontal analysis and zonal elution, 39,40 this method does not need to saturate the binding sites of the immobilized CysLTR1 column, thus it is time-and less labor-intensive.When using this method to explore the binding parameters of the given drug, we usually assume that the binding of the drug to the immobilized receptor occurs rapidly.The adsorption sites are homogeneously distributed on the surface of the stationary phase, while the longitudinal diffusion can be ignored.Therefore, we can obtain the association constants from eqn (1) using the relationship between the molar amount of the injected solute and its capacity factor: k 0 capacity factor of the tested drugs on the immobilized CysLTR1 column.n b , K A , and V m stand for the molar amount of one injection, the association constant, and the void volume of the chromatographic system.n a denotes the total amount of the binding sites of the drug on the column.From eqn (1), a linear regression relationship between k 0 n b 1 þ k 0 and k 0 appears.Thus, we can obtain the association constant and the number of binding sites through the slope and the intercept of the linear relationship.In this set of experiments, we injected a series of concentrations of drugs into the column containing immobilized CysLTR1.Representative chromatograms of the four drugs with different concentrations of each drug injected into the immobilized CysLTR1 column are presented in Fig. 5 under the proposed chromatographic conditions.We found that though the concentration of each drug on the immobilized CysLTR1 column varied, the chromatograms were all similar with a tailing peak.Meanwhile, the retention time of each drug decreased when the injection concentration of the drug increased.The variations in the capacity factors of each drug negatively corresponded to the increased concentration of the drug.This phenomenon is reasonable as the active binding sites of the immobilized CysLTR1 in the column are constant.With the increasing concentration of the injected drugs, a decreased retention time was observed on the column.This result agrees well with the assumption of the injection amount-dependent method.Thus, using eqn (1), the linear relationships between k 0 n b /(1 + k 0 ) versus k 0 V m for the four drugs are depicted in Fig. 6.Based on the equations, the association constant, and the number of binding sites are calculated to be 2.193 × 10 5 L mol −1 , 2.299 × 10 −8 mol L −1 for zarlukast, 4.789 × 10 5 L mol −1 , 2.598 × 10 −8 mol L −1 for pranlukast, 4.272 × 10 5 L mol −1 , 2.094 × 10 −8 mol L −1 for MK-571, and 7.268 × 10 5 L mol −1 , 1.237 × 10 −8 mol L −1 for RA, respectively.The order in which the association constants of the three ligands could be ranked for the immobilized CysLTR1 is pranlukast > MK-571 > zarlukast, which agrees well with the references.Thus, we believe that the established immobilized CysLTR1 chromatography can be used for exploring the binding parameters of drugs.

Conclusions
Herein, in this work, we predicted and investigated the potential binding of rosmarinic acid to CysLTR1 using molecular docking and injection amount-dependent methods.It was found that Phe 158, Tyr 108, Ser 155, Leu 189, His 190, Arg 253, His 256, Pro 176, Val 277, Thr 100, and Leu 281 on CysLTR1 were the main amino acid residues that participated in the binding of drugs to the receptor.The feasibility of the immobilized CysLTR1 column was investigated by injection-amount dependent method to explore the binding parameters of the four drugs.The results reveal that it has the potential to become a powerful tool for the design and screening of important compounds that specically bind to the target protein by immobilized receptor chromatography.

Fig. 1
Fig. 1 Molecular docking analysis of the binding interaction between the four drugs and CysLTR1.(A1-D1), chemical structures of rosmarinic acid, pranlukast, zafirlukast and MK-571.(A2-D2) and (A3-D3) were 3D and 2D overviews of the binding interactions between the four drugs and CysLTR1 by molecular docking.The receptor was shown as ribbon and the drugs were displayed as spheres.